Our research is directed toward elucidation of basic mechanisms involved in the production of cellular damage during exposure to oxidative stress and the contributions of such damage to aging and disease. Our current research involves studies in the following areas of research: (a) Regulation of apoptosis. Caspases, a family of cysteine proteases, play an important role in many forms of cell death by apoptosis and in proinflammatory cytokine maturation. To date, 14 caspases have been identified in mammalian cells. In the present study we used both murine L929r and L929s fibrosarcuma cells to examine the effect of IFN-gamma on caspase-12 gene expression. Cells were first incubated with or without IFN-gamma for 24 hours and caspase-12 mRNA and protein were monitored using semiquantitative RT-PCR and western blot analysis, respectively. We found that IFN-gamma induces caspase-12 expression at both mRNA and protein levels. In addition, IFN-gamma-induced caspase-12 was concentration and time dependent with a substantial induction observed as early as 1 hour after cell stimulation. IFN-gamma-induced caspase-12 was also observed in cells lacking p53. Our results showed that IFN-gamma-induced caspase-12 accumulation was not inhibited by cycloheximide, suggesting that ongoing protein synthesis is not necessary for the induction. However, actinomycin D strongly blocked IFN-gamma-induced caspase-12, providing evidence of transcriptional control. To clarify whether a posttranscriptional mechanism may be involved, the half-life of caspase-12 mRNA was examined in cells treated with actinomycin D alone or with both actinomycin D and IFN-gamma, and caspase-12 mRNA was monitored over a period of time. We did not observe any difference in the caspase-12 mRNA decay under either situation. Since transcriptional activation of IFN-gamma-induced genes is mediated by Janus kinase (JAK) pathway, we next examined whether JAK mediates IFN-gamma-induced caspase-12 expression. We found that cell pretreatment with JAK inhibitor, AG-490, blocked the accumulation of caspase-12 mRNA induced by IFN-gamma. (b) Role of apoptosis in aging. To test the possibility that an age-dependent loss in ability to mediate apoptosis might contribute to the increase in level of oxidized proteins as is known to occur during aging, cultured human fibroblasts from individuals of different ages (17-80 years old) were examined for their ability to induce apoptosis in the presence of hydrogen peroxide. It was confirmed that the levels of oxidatively modified proteins increases with age, not only in whole lysates but also in the mitochondrial fractions, and that these changes correlated with a decline in the intracellular level of ATP. The cells from young individuals (<60 years old) were more resistant than cells from older individuals (>60 years old) to necrotic cell death induced by hydrogen peroxide, and levels of ATP in the old cells was lower than in the young cells. Treatment of cells at all ages with inhibitors of ATP synthesis (oligomycin, 2-deoxyglucose, or 2,4-dinitrophenol) made them more susceptible to cell death, and led also to a switch in the death mode from apoptosis to necrosis. Furthermore, hydrogen peroxide treatment led to a greater release of several inflammatory cytokines (IL-6. I:-7, IL-16, IL-17) in cultures of old cells than in cultures of young cells. Collectively, these results suggest that an age-related decline in the intracellular ATP level reduces the capacity to induce apoptosis and promotes necrotic inflammation. This switch may trigger a number of age-related disorders. (c) Examination of apoptosis in unicellular organisms. To determine the effects of acute oxidative stress on apoptosis in yeast, we carried out studies with Saccharomyces cerevisiae, which contains a gene (YCA1) that encodes synthesis of metacaspase, a homolog of mammalian caspase, and is known to play a crucial role in the regulation of yeast apoptosis. We found that exposure of this yeast strain to hydrogen peroxide led to oxidation of intracellular proteins to carbonyl and methionine sulfoxide derivatives, and that these oxidations were much greater in the strain lacking YCA1 than in the wild type strain. We showed further that exposure of the mutant strain to hydrogen peroxide leads to dramatic up-regulation of the 20S proteasome and to a decrease in the level of ubiquitinylated proteins. (d) Effect of bicarbonate-carbon dioxide buffers on iron-catalyzed oxidation of low density lipoprotein. It is well established that oxidation of low density lipoproteins (LDL) is implicated in atherosclerosis. Although many studies have been carried out to establish mechanisms involved in LDL oxidation, almost all of these studies have been carried out in non-physiological buffer systems. Recently, we showed that metal-catalyzed oxidation of LDL to malondialdehyde, protein carbonyl, and methionine sulfoxide derivatives is much greater when reactions are carried out in physiological bicarbonate buffer systems. In continuing studies, it was shown that hemin or heme-containing proteins (hemoglobin or cytochrome c) could replace free metal ions in hydrogen peroxide-facilitated oxidation of LDL when the oxidations were carried out in bicarbonate buffers. We found also that bicarbonate buffer enhanced the oxidation of LDL by the cytochrome P450 system. Results of these studies suggest that bicarbonate radicals and/or peroxycarbonate might be implicated in LDL oxidation under physiological conditions. (e) Cyclic nitration and denitrification of tyrosine residues of proteins. Other investigators have reported that the nitro-groups of nitrotyrosine residues can be removed by an as yet undetermined mechanism. Significantly, the removal of nitro-groups provides a mechanism for the repair of nitration damage. But, in theory, the cyclic nitration and denitrification of tyrosine could also provide a mechanism for the scavenging of peroxynitrite. Prompted by this consideration, we have initiated a research effort designed to confirm the existence of a biological system for the denitrification of 3-nitrotyrosine derivatives, and the possibility that such a system can scavenge peroxynitrite. To achieve our goal, we are carrying out studies with: (1) mammalian RAW 264.7 cells; (2) an aerobic bacterium (pseudomonas JS51 strain) that can use nitrobenzoic acid as a source of nitrogen for growth; and (3) an anaerobic bacterium that can use 3-nitrophenol as the sole source of nitrogen for growth. So far, we have developed a HPLC method to quantify tyrosine, nitrotyrosine, and aminotyrosine in protein samples, which will be used to determine the fate of the nitro-compounds in our samples. (f) Biochemical consequences of RNA oxidation. In continuing studies on the oxidative modification of mRNA, we examined the effects of oxidation by mixed-function oxidation systems composed of either ferrous iron/ascorbate/hydrogen peroxide (Fenton system) or cytochrome c/hydrogen peroxide. Oxidation by the cytochrome c system led to a selective decrease in the guanosine ribonucleotide content of the RNA, whereas oxidation by the Fenton system led to a decrease in all types of ribonucleotides. The oxidation of RNA by cytochrome c was also associated with an increase in molecular weight of the RNA, likely due to covalent attachment of cytochrome c. Interestingly, the oxidation of guanosine residues by the cytochrome system was substantially greater when the oxidation was carried out in the physiological bicarbonate buffer. Collectively, results of these studies indicate that oxidation of mRNA leads to suppression of its translation ability with respect to both initiation and elongation and/or to mutational errors of reverse transcription.